Normal bone formation is a dynamic process that occurs throughout life for the development, growth, and maintenance of the skeleton with its important properties such as stability, strength, and resistance to fracture. It is of critical importance in bone disorders and bone injuries which afflict millions of children and adults yearly. Much of the process and factors involved in bone formation and its regulation still remain poorly understood, which contributes to important gaps in understanding the underlying causes of bone disorders, and approaches for prevention and treatment. It has been known for ~70 years that bone (and teeth) in all vertebrates contains extremely high level of citrate (100-fold greater than most soft tissues). Yet, the role, source, and factors involved in its production and regulation still remain largely unknown; and the implications of citrate in bone have been essentially ignored and absent over the past 35 years. Recently, citrate has been discovered to be a major component of the apatite nanocrystal structure; and is essential for the important properties of bone. Thus, an indispensable role of citrate in bone formation is now re-established. Our broad, long-term objectives are to identify the source and factors involved in bone citrate production and regulation. This proposal begins to address the essential question What is the source of citrate in bone? This R21 proposal explores our innovative hypotheses that: The osteoblasts are 'specialized citrate-producing' cells that provide the source of citrate in bone formation. This functional/metabolic capability occurs during the osteogenic differentiation process of the mesenchymal stem cells in order to provide osteoblast 'citration' in concert with mineralization for normal bone formation. Since high zinc levels accompany the high citrate levels in bone, the hypothesis includes the new concept that zinc is required for the capability of osteoblast citrate production. The specific aims will employ the osteogenic differentiation of mesenchymal stem cells to osteoblasts: 1) to establish that differentiated osteoblasts will exhibit the capability of net citrate production under appropriate conditions; and to determine when during the differentiation stages, citrate production (citration process) occurs in relation to the mineralization process; 2) to determine the conditions required to facilitate and optimize osteoblast citrate production: e.g. the requirements for vitamin D vs. dexamethasone. The study will show for the first time that osteoblasts are specialized zinc-accumulating citrate-producing cells that provide the source of citrate in bone formation. The success of this exploratory study will permit studies to resolve the other important issues and questions such as how is osteoblast citrate production synchronized with its incorporation into apatite nanocrystal formation; how and when are zinc and zinc transporters up- regulated in concert with citrate production; and clinical issues such as: What are the implications of citrate relationships in different bone disorders, fractures and repair; what new targets exist for agents employed in treatment of bone disorders? A new direction and insight into osteoblast role and bone formation will evolve.